Major depressive disorder (MDD) is a devastating psychiatric disorder that affects millions of Americans. Despite substantial research, no specific risk factor has yet been identified as having a causal role in MDD. Epigenetic modifications, especially DNA methylation, are increasingly being recognized as a key mechanism involved in the pathogenesis of depression. However, the biological pathways linking aberrant methylation to depression remain poorly understood. This uncertainty greatly hampers our ability to implement early diagnosis, prevention and treatment for this debilitating disorder. The objective of this study is to identify functional epigenetic determinants for MDD. Our central hypothesis is that aberrant DNA methylation and resulting alterations in gene expression are associated with MDD. The rationale for the proposed research is that: once we know the epigenetic determinants for depression, we will be able to develop novel epigenetic markers and therapeutic targets for risk assessment, prevention and treatment of MDD and related psychiatric conditions. We proposed three specific aims: (1) Identify differentially methylated regions (DMRs) associated with MDD. This aim is to conduct an epigenome-wide DNA methylation analysis to identify epigenetic variations contributing to MDD in monocytes DNA from 100 monozygotic (MZ) discordant twin pairs from the University of Washington Twin Registry (UWTR), a large community-based twin registry in the U.S. (2) Replicate the top 50 ranked genes from Aim 1 in two independent samples, including 80 MZ discordant twin pairs recruited from the same registry and 36 postmortem brain tissue of well-characterized MDD patients and matched controls. (3) Determine the functional importance of the positive methylation findings in both blood and brain by profiling gene expression levels in each of the four brain regions (frontal cortex, hippocampus, amygdala, and cingulate cortex). Differential expressed genes related to MDD will be identified. Integrative analyses will be performed to elucidate the connections between DNA methylation patterns and gene expression of cognate genes in relation to MDD. The proposed study is the only one of its kind to identify functional epigenetic determinants for MDD in a well- matched MZ discordant twin sample, followed by replication in postmortem brain tissue, the affected organ in MDD. The work proposed here is expected to have an important positive impact, because genes with both differential methylation and expression are highly likely to provide novel epigenetic targets for prevention, intervention and treatment for depression and its related psychiatric conditions in addition to fundamentally advancing the fields of psychiatric genetics.